GSM7011610: Epithelial cells AT2- and ciliated-depleted and dividing epithelial cells, SfSRC mice, uninjured lung, day0; Mus musculus; RNA-Seq
PBS-perfused lungs were inflated with 2 ml of digestion cocktail containing 50 U/ml dispase (Corning), 250 U/ml collagenase type I (Worthington), 5 U/ml elastase (Worthington), and 60 U/ml DNAse I (Roche). Trachea was clipped distally and lungs were dissected in a petri dish on ice to remove extrapulmonary airways (trachea and main bronchi). Lung lobes were placed in a C tube (Miltenyi) containing 3 ml of digestion cocktail, and the m_lung_01 program was run on gentleMACS (Miltenyi). C tubes were placed in a rotating incubation oven at 37ºC for 30 minutes. The m_lung_02 program was run again, and the tubes were placed on ice for the next steps. The lung cells were passed through a 70 µm cell strainer (Corning) and centrifuged at 400 g for 5 minutes. The pellet was resuspended in a red blood lysis buffer solution (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubated for 2 minutes on ice, and washed with EasySep buffer (STEMCELL Technologies) at 400 g for 5 minutes. To isolate mesenchymal cells, a milder digestion cocktail was used containing only 375 U/ml collagenase and 60 U/ml DNase I. Libraries were prepared using Chromium Next GEM Single Cell 3' v2 (samples 1 to11) or Chromium Next GEM Single Cell 3' v3.1 (samples 12 to16) and following the manufacturer's protocol (10x Genomics)